Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila.
A new method is described for cloning DNA sequences occupied by a specific protein on chromatin in vivo . The approach uses UV cross-linking to couple proteins covalently to DNA and the resulting complexes are then purified under stringent conditions. Particular adducts are immunoprocipitated with antibody to the protein of interest. The resulting DNA (iDNA) is amplified by PCR, cloned and characterized. The model system used was RNA polymerase II (Pol II), whose density on particular DNAs under various conditions is well documented. Pol II can exist in several states on DNA. While Pol II can simply be bound to DNA, the bulk of DNA-associated Pol II is transcriptionally engaged in either the transcribing or paused states. Paused Pol IIs that have previously been characterized are found at promoters and have the distinctive property that their transcription in isolated nuclei is stimulated by sarkosyl or high salt. Here we isolate and sequence DNAs that cross-link to Pol II molecules. We identify by nuclear run-on assays those DNAs that have Pol II engaged in transcription. Twenty one percent of the iDNA clones that have detectable transcriptionally engaged Pol II appear to be paused, in that they display sarkosyl-stimulated trancription in a nuclear run-on transcription assay. At least some of these map to the 5'-ends of genes. These results suggest that transcriptional pausing of Pol II is a general phenomenon in vivo.
Law
A
A
Section of Biochemsitry, Molecular and Cellular Biology, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.
Hirayoshi
K
K
O'Brien
T
T
Lis
J T
JT
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D017344:Genes, Insect; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004247:DNA; D012319:RNA Polymerase II